1. Field of the Invention
The present invention relates to a modified thermostable DNA polymerase, a DNA polymerase composition for amplifying nucleic acid, and a reagent for amplifying nucleic acid containing said enzyme or composition, as well as a method for amplifying nucleic acid by use of said reagent.
2. Description of the Related Art
Conventionally, a large number of studies have been conducted on thermostable DNA polymerases for use in techniques for amplification of nucleic acid, such as polymerase chain reaction (PCR) etc. Examples of thermostable DNA polymerases used in PCR are DNA polymerase (Tth polymerase) mostly derived from Thermus thermophilus and DNA polymerase (Taq polymerase) derived from Thermus aquaticus. Other known examples are DNA polymerases derived from a hyperthermophilic archaeon strain, such as thermostable DNA polymerase derived from Pyrococcus furiosus (Pfu polymerase, WO92/09689, Unexamined Published Japanese Patent Application No. 328,969/1993) and thermostable DNA polymerase derived from Thermococcus litoralis (Tli polymerase, Unexamined Published Japanese Patent Application No. 7160/1994).
The present inventors have previously found thermostable DNA polymerase excellent in thermostability and DNA extension rate derived from Pyrococcus sp. KOD1 (KOD polymerase, Unexamined Published Japanese Patent Application No. 298,879/1995).
However, these thermostable DNA polymerases have problems such as insufficient amplification of nucleic acid. Further problems with polymerase derived from a hyperthermophilic archaeon strain such as Pyrococcus sp. KOD1. are that it has a 3'-5' exonuclease activity and there is a limit to PCR conditions including reaction time, enzyme amount, primer concentration etc. Therefore, there is demand for novel thermostable DNA polymerase.
As a result of their eager research, the present inventors have succeeded in creating a modified enzyme derived from Pyrococcus sp. KOD1, said enzyme having the 3'-5' exonuclease activity reduced to 5% or less of the original polymerase before modification while maintaining the DNA extension rate and thermostability of the original polymerase.
The present inventors have further found that the efficiency of amplification using a polymerase before modification is improved by use of its modified thermostable DNA polymerase having a DNA extension rate of at least 30 bases/second and capable of maintaining 60% or more residual activity at pH 8.8 (determined at 25.degree. C. because it was difficult to measure the pH at 95.degree. C.) after treatment at 95.degree. C. for 6 hours, said modified enzyme having the 3'-5' exonuclease activity reduced to 5% or less of the polymerase before modification, and the present inventors thereby completed the present invention.
That is, the present invention is a modified thermostable DNA polymerase having the following physicochemical properties:
action: it has a DNA polymerase activity and has 5% or less of the 3'-5' exonuclease activity of the enzyme before modification; PA1 DNA extension rate: at least 30 bases/second; and PA1 thermostability: it is capable of maintaining 60% or more residual activity at pH 8.8 (determined at 25.degree. C.) after treatment at 95.degree. C. for 6 hours.
Further, the present invention is a method for amplifying nucleic acid, which comprises reacting DNA as a template, primers, dNTP and the thermostable DNA polymerase of claims 1 to 3, thus extending the primers to synthesize DNA primer extension products.
Further, the present invention is a reagent for amplifying nucleic acid, which comprises 2 kinds of primer, one of which is complementary to a DNA extension product of another primer, dNTP, said thermostable DNA polymerase, and a buffer solution.
As one of methods for amplifying long chain nucleic acid, there is a report on PCR making use of both Taq polymerase (KlenTaq-278) free of 3'-5' exonuclease activity and Pfu polymerase (or Tli polymerase) having 3'-5' exonuclease activity, or of a DNA polymerase composition containing a mixture of their mutant enzymes (Barns, W. M. (1994) Proc. Natl. Acad. Sci. USA 91, 2216-2220).
There is another report on PCR making use of a polymerase composition containing a mixture of Tth polymerase free of 3'-5' exonuclease activity, Pfu polymerase (or Tli polymerase) with 3'-5' exonuclease activity, and thermostable DNA polymerase derived from Thermotoga maritima (Unexamined Published Japanese Patent Application No. 38198/1996).
Higher efficiency of amplification can be attained by such a composition than by one kind of DNA polymerase but is still not sufficient because 2 kinds of DNA polymerase having different properties in thermostability and DNA extension rate are used. Hence, there has been demand for a method further excellent in efficiency of amplification.
As a result of their eager research under these circumstances, the present inventors found that PCR excellent in efficiency of amplification can be effected using a DNA polymerase composition for nucleic acid amplification, consisting of a combination of first and second DNA polymerases being almost identical to each other with respect to thermostability and DNA extension rate, the activity of the second DNA polymerase being present at a lower level than that of the first DNA polymerase, specifically a DNA polymerase composition comprising DNA polymerases selected from the group consisting of a modified thermostable DNA polymerase (first polymerase) having 0 to 5% of the 3'-5' exonuclease activity of the naturally occurring enzyme before modification and a modified thermostable DNA polymerase (second polymerase) having 100 to 6% of the 3'-5' exonuclease activity of a naturally occurring DNA polymerase or of its original naturally occurring enzyme before modification.
That is, the present invention is a DNA polymerase composition for amplifying nucleic acid, which comprises a modified thermostable DNA polymerase having 0 to 5% of the 3'-5' exonuclease activity of the original enzyme before modification (first polymerase) and the original enzyme or a modified thermostable DNA polymerase having 100 to 6% of the 3'-5' exonuclease activity of its original thermostable enzyme before modification (second polymerase), said first and second DNA polymerases having a DNA extension rate of at least 30 bases/second and capable of maintaining 60% or more residual activity at pH 8.8 (determined at 25.degree. C.) after treatment at 95.degree. C. for 6 hours.
Further, the present invention is a method for amplifying nucleic acid, which comprises reacting DNA as a template, primers, dNTP and said DNA polymerase composition, thus extending the primers to synthesize a DNA primer extension product.
Further, the present invention is a reagent for amplifying nucleic acid, which comprises 2 kinds of primer, one of which is complementary to a DNA extension product of another primer, dNTP, said DNA polymerase composition, divalent ions, monovalent ions, and a buffer solution.